Reconstruction of urological structures with polymeric matrices

ABSTRACT

A method for repairing defects and reconstructing urothelial structures in vivo has been developed using a fibrous, open synthetic, biodegradable polymeric matrix which is configured to provide the desired corrective structure. The matrix is shaped to correct the defect, then implanted surgically to form a scaffolding for the patient&#39;s own cells to grow onto and into. The implantation of the matrix initiates an inflammatory reaction, resulting in urothelial cells, endothelial cells and mesenchymal cells, to migrate into the matrix. The polymer forming the matrix is selected to be biocompatible and degradable in a controlled manner over a period of one to six months, in the preferred embodiment. A preferred material is a poly(lactic acid-glycolic acid) in a fibrous form, such as a woven or non-woven mesh. Examples demonstrate the repair of defects in bladder in rabbits.

BACKGROUND OF THE INVENTION

[0001] The present invention is generally in the area of methods forreconstruction of urothelial structures, especially bladders.

[0002] Traditionally, defects in the bladder and other urothelialstructures have been corrected surgically. This has obviousdisadvantages when there is a defect in the structure which requiresclosure of an opening for which there is insufficient tissue or when thestructure itself is deformed or too small to meet the needs of thepatient.

[0003] Bowel segments have been used in reconstruction of genitourinarystructures in these circumstance. The use of bowel in genitourinaryreconstruction is associated with a variety of complications, includingmetabolic abnormalities, infection, perforation, urolithiasis, increasedmucus production and malignancy, as reviewed by Atala, A. and Retik, A.:Pediatric urology—future perspectives. In: Clinical Urology. Edited byR/J. Krane, M. B. Siroky and J. M. Fitzpatrick. (Philadelphia: J. B.Lippincott, 1993). Alternative approaches need to be developed toovercome the problems associated with the incorporation of intestinalsegments into the urinary tract. Natural tissues and synthetic materialsthat have been tried previously in experimental and clinical settingsinclude omentum, peritoneum, seromuscular grafts, de-epithelializedsegments of bowel, polyvinyl sponge and polytetrafluoroethylene(Teflon). These attempts have usually failed.

[0004] It is evident that urothelial-to-urothelial anastomoses arepreferable functionally. However, the limited amount of autologousurothelial tissue for reconstruction generally precludes this option. Incell transplantation, donor tissue is dissociated into individual cellsor small tissue fragments and either implanted directly into theautologous host or attached to a support matrix, expanded in culture andreimplanted after expansion. Autologous skin cells have been used inthis fashion in the treatment of extensive burn wounds, as reported byGreen, et al., “Growth of cultured human epidermal cells into multipleepithelia suitable for grafting”, Proc. Natl. Acad. Sci., 76:5665(1979); O'Connor, et al., “Grafting of burns with culture epitheliumprepared from autologous epidermal cells”, Lancet, 1:75 (1981); andBurke, et al., “Successful use of a physiologically acceptableartificial skin in the treatment of an extensive burn injury”, Ann.Surg., 194:413 (1981).

[0005] A suitable material for a cell transplantation matrix must bebiocompatible to preclude migration and immunological complications, andshould be able to support extensive cell growth and differentiated cellfunction. It must also be resorbable, allowing for a completely naturaltissue replacement. The matrix should be configurable into a variety ofshapes and should have sufficient strength to prevent collapse uponimplantation. Recent studies indicate that the Biodegradable polyesterpolymers made of polyglycolic acid seem to fulfill all of thesecriteria, as described by Vacanti, et al., “Selective celltransplantation using bioabsorbable artificial polymers as matrices”, J.Ped. Surg., 23:3 (1988); Cima, et al., “Hepatocyte culture onbiodegradable polymeric substrates”, Biotechnol. Bioeng., 38:145 (1991);Vacanti, et al., “Synthetic polymers seeded with chondrocytes provide atemplate for new cartilage formation”, J. Plast. Reconstr. Surg., 88:753(1991).

[0006] The feasibility of using biodegradable polymers as deliveryvehicles for urothelial cell transplantation has been demonstrated bystudies showing that urothelial cells will adhere to synthetic polymerscomposed of polyglycolic acid and survive in vivo, as reported by Atala,et al., “Formation of urothelial structures in vivo from dissociatedcells attached to biodegradable polymer scaffolds in vivo”, J. Urol.,part 1, 148:658 (1992).

[0007] For implantation of cells on polymer matrices to be successful inpatients, a source of an effective concentration of cells has to beavailable, and the urothelial cell population has to survive forextended times on implanted polymers and proliferate extensively invivo. Most importantly, implanted cells have to remain intact as definedstructures as the polymer implant degrades over time under physiologicalconditions. Polymer scaffolds would have to include bladder smoothmuscle in concert with urothelial cells to reconstitute a functionalbladder wall.

[0008] An easier solution would be to develop a method for correctingdefects which did not require obtaining and implanting cells on thepolymer matrices. However, initial studies with chondrocytes implantedin tissue in the absence of a matrix and implantation of polymer alonehas not been demonstrated to result in appropriate ingrowth andproliferation of cells.

[0009] It is therefore an object of the present invention to provide amethod and means for reconstructing defects in organ structures,especially urothelial structures such as the bladder, ureter andurethra, which does not require exogenous cells.

SUMMARY OF THE INVENTION

[0010] A method for repairing defects and reconstructing urologicalstructures in vivo has been developed using a fibrous, open, synthetic,biodegradable polymeric matrix. The matrix is shaped to correct thedefect, then implanted surgically to form a scaffolding for the patientsown cells to grow onto and into. The implantation of the matrixinitiates an inflammatory reaction, resulting in urothelial cells,including both endothelial cells and mesenchymal cells, migrating intothe matrix. The polymer forming the matrix is selected to bebiocompatible and degradable in a controlled manner over a period of oneto six months in the preferred embodiment. A preferred material is apolyhydroxy acid, poly(lactic acid-glycolic acid), in a fibrous form,such as a woven or non-woven mesh.

[0011] Examples demonstrate the repair of defects in bladders inrabbits.

DETAILED DESCRIPTION OF THE INVENTION

[0012] Previous studies have indicated that cells implanted in theabsence of a matrix and that matrices implanted in the absence of seededcells do not form structures. In contrast, previous studies haveindicated that very small repairs can be achieved by covering the defectwith a “patch” or other biodegradable or non-degradable mesh, so thatthe surrounding tissue grows over the defect. The usefulness ofpolymeric matrices, in the absence of seeded cells, either before orafter implantation of the matrix, to form tissue structures, issurprising. Based on the previous studies, one would have expectedproblems, including compression of the matrix after surgical attachmentwhich would prevent cells from entering into and proliferating in thematrix to form tissue; migration into and proliferation within thematrix of the wrong cell populations; and/or that the matrix would havedetached or degraded prior to tissue formation. As demonstrated by thefollowing examples, none of these problems occurred and the materialsdid form tissue that effectively repaired the defects in bladders.

[0013] Polymeric Materials

[0014] A variety of polymeric materials can be used to make the matrix.In the preferred embodiment, the material is biocompatible,biodegradable over a period of one to six months, synthetic, and easilyfabricated. The most preferred material is poly(lactic acid-glycolicacid).

[0015] In the preferred embodiment, the matrix is formed of abioabsorbable, or biodegradable, synthetic polymer such as apolyanhydride, polyorthoester, polyhydroxy acid, for example, polylacticacid, polyglycolic acid, and copolymers or blends thereof, andpolyphosphazenes. Collagen can also be used, but is not as controllableas a synthetic polymer either with respect to manufacture of matrices ordegradation in vivo and is therefore not preferred. These materials areall commercially available.

[0016] In some embodiments, attachment of the cells to the polymer isenhanced by coating the polymers with compounds such as basementmembrane components, agar, agarose, gelatin, gum arabic, collagens typesI, II, III, IV and V, fibronectin, laminin, glycosaminoglycans, mixturesthereof, and other materials known to those skilled in the art of cellculture.

[0017] All polymers for use in the matrix must meet the mechanical andbiochemical parameters necessary to provide adequate support for thecells with subsequent growth and proliferation. The polymers can becharacterized with respect to mechanical properties such as tensilestrength using an Instron tester, for polymer molecular weight by gelpermeation chromatography (GPC), glass transition temperature bydifferential scanning calorimetry (DSC) and bond structure by infrared(IR) spectroscopy, with respect to toxicology by initial screening testsinvolving Ames assays and in vitro teratogenicity assays, andimplantation studies in animals for immunogenicity, inflammation,release and degradation studies.

[0018] One of the advantages of a biodegradable polymeric matrix is thatangiogenic and other bioactive compounds can be incorporated directlyinto the matrix so that they are slowly released as the matrix degradesin vivo. As the cell-polymer structure is vascularized and the structuredegrades, the cells will differentiate according to their inherentcharacteristics. Factors, including nutrients, growth factors, inducersof differentiation or de-differentiation (i.e., causing differentiatedcells to lose characteristics of differentiation and acquirecharacteristics such as proliferation and more general function),products of secretion, immunomodulators, inhibitors of inflammation,regression factors, biologically active compounds which enhance or allowingrowth of the lymphatic network or nerve fibers, hyaluronic acid, anddrugs, which are known to those skilled in the art and commerciallyavailable with instructions as to what constitutes an effective amount,from suppliers such as Collaborative Research, Sigma Chemical Co.,vascular growth factors such as vascular endothelial growth factor(VEGF), EGF, and HB-EGF, could be incorporated into the matrix orprovided in conjunction with the matrix. Similarly, polymers containingpeptides such as the attachment peptide RGD (Arg-Gly-Asp) can besynthesized for use in forming matrices.

[0019] A presently preferred polymer is polyglactin 910, developed asabsorbable synthetic suture material, a 90:10 copolymer of glycolide andlactide, manufactured as Vicryl® braided absorbable suture (Ethicon,Inc., Somerville, New Jersey) (Craig, P. H., Williams, J. A., Davis K.W., et al.: A Biological Comparison of Polyglactin 910 and PolyglycolicAcid Synthetic Absorbable Sutures. Surg., 141:1010 (1975). Acommercially available surgical mesh formed of polyglycolic acid,Dexon™, can also be used.

[0020] Matrix Design

[0021] The design and construction of the scaffolding is of primaryimportance. The matrix should be a pliable, non-toxic, injectable poroustemplate for vascular ingrowth. The pores should allow vascularingrowth. These are generally interconnected pores in the range ofbetween approximately 100 and 300 microns, i.e., having an interstitialspacing between 100 and 300 microns, although larger openings can beused. The matrix should be shaped to maximize surface area, to allowadequate diffusion of nutrients, gases and growth factors to the cellson the interior of the matrix and to allow the ingrowth of new bloodvessels and connective tissue. At the present time, a porous structurethat is relatively resistant to compression is preferred, although ithas been demonstrated that even if one or two of the typically six sidesof the matrix are compressed, that the matrix is still effective toyield tissue growth.

[0022] Fibers (sutures or non-woven meshes) can be used as supplied bythe manufacturer. Other shapes can be fabricated using one of thefollowing methods:

[0023] Solvent Casting. A solution of polymer in an appropriate solvent,such as methylene chloride, is cast on a fibrous pattern reliefstructure. After solvent evaporation, a thin film is obtained.

[0024] Compression Molding. Polymer is pressed (30,000 psi) into anappropriate pattern.

[0025] Filament Drawing. Filaments are drawn from the molten polymer.

[0026] Meshing. A mesh is formed by compressing fibers into a felt-likematerial.

[0027] At the present time, a mesh-like structure formed of fibers whichmay be round, scalloped, flattened, star shaped, solitary or entwinedwith other fibers is preferred. As discussed above, the polymeric matrixmay be made flexible or rigid, depending on the desired final form,structure and function. either woven, non-woven or knitted material canbe used. A material such as a velour is an example of a suitable wovenmaterial. The fibers can be fused together by addition of a solvent ormelting to form a more stable structure. Alternatively, high pressurejets of water onto a fibrous mat can be used to entangle the fibers toform a more rigid structure. For repair of a defect, for example, aflexible fibrous mat is cut to approximate the entire defect, thenfitted to the surgically prepared defect as necessary duringimplantation. An advantage of using the fibrous matrices is the ease inreshaping and rearranging the structures at the time of implantation.

[0028] A sponge-like structure can also be used. The structure should bean open cell sponge, one containing voids interconnected with thesurface of the structure, to allow adequate surfaces of attachment forsufficient cells to form a viable, functional implant.

[0029] Implantation of the Matrix

[0030] The matrix is implanted using standard surgical procedures,suturing edges to the tissue to be treated or adjacent materials asnecessary.

[0031] This method of using a polymer as a scaffold wherein adjacentcells can migrate onto and into the polymer can be used to patch defectsof urethelial associated organs such as urethra, bladder, ureters, andrenal pelvis. In addition, this method can be used to entirely replaceor reconstruct these structures, such as for hypospadias, where urethralreconstructive surgery is necessary, or for bladder surgery where eitheran augmentation is necessary for a low capacity bladder or a neobladderis needed, or for ureteral extension, replacement, or reconstruction,such as with a patient requiring additional ureteral length secondary totrauma or neoplasm. Further, this system can be used for other areaswhere a soft tissue replacement is needed such as in thegastrointestinal system, for example, in situations where additionalintestinal tissue is needed, or in the musculoskeletal system, such asfor bone or cartilage tissue replacement secondary to congenital,neoplastic, inflammatory, or traumatic conditions.

[0032] The present invention will be further understood by reference tothe following non-limiting examples.

EXAMPLE 1 Urethral Reconstruction Using Biodegradable Polymer Scaffolds

[0033] The more severe forms of hypospadias are usually corrected with avascularized preputial island graft. Patients with failedreconstruction, epispadias, or urethral strictures may not havesufficient preputial skin for repair. In these instances, severalalternatives have been used, including free skin, bladder mucosa andbuccal mucosa grafts. However, some of these grafts are associated withseveral complications, and their use is limited. The following studycompares the usefulness of cell-polymer matrices and synthetic polymermatrices in the absence of seeded cells for repair of urothelialstructures, especially bladder.

[0034] Materials and Methods

[0035] Urothelial cells were harvested from a small segment of thebladders of 10 New Zealand white rabbits by open surgery. The urothelialcells were plated in vitro, expanded, and tagged with 7-amino4-chloromethylcoumarin, a fluorescent probe. Cells were resuspended inmedia and seeded onto biodegradable polymer scaffolds. Partialurethrectomies were performed in each rabbit through a circumcisingincision. The autologous urothelial cell-polymer meshes were interposedusing continuous 7-0 Vycril™ sutures to form the neourethras. Polymermeshes without urothelial cells were used in two animal as controls. Thepenile skin was closed over the neourethra with interrupted 5-0 Vycril™sutures. Due to the thick, semi-solid consistency of rabbit urine,simultaneous vesicotomies were performed in order to achieve asatisfactory urinary diversion.

[0036] After vesicotomy closure, ten days after urethral reconstruction,the animals were able to void through the neourethra withoutcomplications. Retrograde urethrograms showed no evidence of strictureformation. Histologic examination of the neourethras demonstratedcomplete re-epithelialization of the polymer mesh sites by day 14. Thesefindings were persistent at the four and six week time points.Fluorescent microscopy showed tagged autologous urothelial cells closelyassociated with the poly fibers. Urethral polymer controls showedcomplete re-epithelialization, by 14 days, indicating that native cellsare not necessary for successful replacement of urethral defects. Thepolymer fibers were partially degraded by day 14 and almost totallyreabsorbed by day 30.

[0037] In conclusion, biodegradable polymer meshes can be used, eitheralone or in combination with harvested autologous urothelial cells, forurethral reconstruction. Adequate anatomic and functional replacementcan be achieved by using this technology.

EXAMPLE 2 Bladder Reconstruction Using Biodegradable Polymer

[0038] Multiple anomalies of the bladder, whether congenital oracquired, require augmentation cystoplasty. In these instances, use ofbowel for augmentation has been used widely. The use of gastrointestinaltissue for urologic reconstruction is associated with severalcomplications, and their use is limited. The following study comparesthe usefulness of cell polymer matrices and cell-polymer matrices andsynthetic polymer matrices in the absence of seeded cells for bladderreconstruction.

[0039] Urothelial cells are harvested from a small segment of thebladders of ten New Zealand white rabbits by open surgery. Theurothelial cells were plated in vitro and tagged with 7-Amino4-chloromethylcomarin, a fluorescent probe. Cells were resuspended inmedia and seeded onto biodegradable polymer scaffolds. Partialcystectomies were performed in each rabbit through a mid-abdominalincision. The autologous urothelial cell-polymer matrices wereinterposed using Vycril™ sutures to augment the small bladders. Polymermatrices without urothelial cells were used in ten additional animals ascontrols. Omentum was used to cover the polymer, rendering itimpermeable to urine. A urethral catheter was left in place for ten daysfor urinary diversion.

[0040] After the catheter was removed, the animals were able to voidwithout complications. Bladder cystograms showed an increased bladdercapacity in all animals. Histological examination of the neobladdersdemonstrated complete re-epithelialization of the polymer mesh sites byday 14. These findings were persistent at the four and six week timepoints. Fluorescent microscopy showed tagged autologous urothelial cellsclosely associated with the polymer fibers. Bladder polymer controlsshowed complete re-epithelialization by 14 days, indicating that nativecells are not necessary for successful bladder augmentation orreconstruction. The polymer fibers were partially degraded by day 14 andalmost totally reabsorbed by day 30.

[0041] In conclusion, biodegradable polymer matrices can be used, eitheralone or in combination with harvested autologous urothelial cells, forbladder reconstruction. Adequate anatomic and functional replacement canbe achieved by using this technology.

EXAMPLE 3 Ureteral Reconstruction Using Biodegradable Polymers Scaffolds

[0042] Multiple anomalies of the ureter, whether congenital or acquired,require ureteral reconstruction. In these instances, when the ureteraltissue present cannot be used for reconstruction, other gastrointestinaltissues have been used. The use of gastrointestinal tissue, however, isassociated with numerous complications when they are interposed with theurinary tract. The following study compares the usefulness of cellpolymer matrices and synthetic polymer matrices in the absence of seededcells for bladder reconstruction.

[0043] Urothelial cells were harvested from a small segment of thebladder of ten beagle dogs by open surgery. The urothelial cells wereplated in vitro, expanded, and tagged with 7-amino4-chloromethylcomarin, fluorescent probe. Cells were resuspended inmedia and seeded onto biodegradable polymer scaffolds. Partialureterectomies were performed in each dog for a flank incision. Theautologous urothelial cell-polymer matrices were interposed usingVicryl™ sutures to interpose these ureters. Polymer matrices withouturothelial cells were used in 10 additional animals as controls.Gerota's fascia was used to cover the polymer, rendering it impermeableto urine. A ureteral catheter was left in place indwelling for ten daysfor urinary diversion.

[0044] After the catheter was removed, an intravenous pyelogram wasperformed which showed normal ureteral anatomy in each animal, withoutany evidence of obstruction. Histological examination of the neo-uretersdemonstrated complete epithelialization of the polymer mesh sites by day14. These findings were persistent at the 4th and 6th week time points.Fluorescent microscopy showed tagged autologous urethelial cells closelyassociated with the polymer fibers. Bladder polymer control showedcomplete re-epithelialization by 14 days, indicating that native cellsare not necessary for successful ureteral reconstruction. The polymerfibers were partially degraded by day 14 and almost totally reabsorbedby day 30.

[0045] In conclusion, biodegradable polymer matrices can be used, eitheralone or in combination with harvested autologous urethelial cells, forureteral reconstruction in large animals.

[0046] Although this invention has been described with reference tospecific embodiments, variations and modifications of the method andmeans for constructing urothelial implants by implantation of polymericmatrices will be apparent to those skilled in the art. Suchmodifications and variations are intended to come within the scope ofthe appended claims.

We claim:
 1. A method for correcting urothelial defects comprisingimplanting at the site to be corrected a matrix of a biodegradable,biocompatible synthetic polymeric fibers forming a matrix having aninterstitial spacing of at least 100 microns, shaped to correct thedefect.
 2. The method of claim 1 wherein the defect is in the bladder.3. The method of claim 1 wherein the defect is in the ureter or urethra.4. The method of claim 1 wherein the defect is in the gastrointestinaltract.
 5. The method of claim 1 further comprising providing with thematrix factors selected from the group consisting of nutrients, growthfactors, inducers of differentiation or dedifferentiation, products ofsecretion, immunomodulators, inhibitors of inflammation, biologicallyactive compounds which enhance or allow ingrowth of the lymphaticnetwork or nerve fibers, vascular growth factors, attachment peptides,and combinations thereof.
 6. The method of claim 1 wherein the matrix iscoated with a material selected from the group consisting of basementmembrane components, agar, agarose, gelatin, gum arabic, collagens typesI, II, III, IV and V, fibronectin, laminin, glycosaminoglycans, andmixtures thereof.
 7. A fibrous matrix formed of biodegradable syntheticpolymer having an interstitial spacing between 100 and 300 microns whichis shaped to repair a tissue defect.
 8. The matrix of claim 7 whereinthe defect is in a urological structure.
 9. The matrix of claim 8 wherethe urological structure is selected from the group consisting ofureters, urethras, bladders and renal pelvis.
 10. The matrix of claim 7wherein synthetic polymer is selected from the group consisting ofpolyanhydrides, polyorthoesters, polyphosphazenes, and polyhydroxyacids.
 11. The matrix of claim 10 wherein the polymer is a polyhydroxyacid is selected from the group consisting of polylactic acid,polyglycolic acid, and copolymers thereof.
 12. The matrix of claim 7wherein the matrix degrades over a period of between one and six monthsfollowing implantation.